The invention relates to compositions and methods useful in the detection of nucleic acids using a variety of amplification techniques, including both signal amplification and target amplification. Detection proceeds through the use of an electron transfer moiety (ETM) that is associated with the nucleic acid, either directly or indirectly, to allow electronic detection of the ETM using an electrode.
The detection of specific nucleic acids is an important tool for diagnostic medicine and molecular biology research. Gene probe assays currently play roles in identifying infectious organisms such as bacteria and viruses, in probing the expression of normal genes and identifying mutant genes such as oncogenes, in typing tissue for compatibility preceding tissue transplantation, in matching tissue or blood samples for forensic medicine, and for exploring homology among genes from different species.
Ideally, a gene probe assay should be sensitive, specific and easily automatable (for a review, see Nickerson, Current Opinion in Biotechnology 4:48-51 (1993)). The requirement for sensitivity (i.e. low detection limits) has been greatly alleviated by the development of the polymerase chain reaction (PCR) and other amplification technologies which allow researchers to amplify exponentially a specific nucleic acid sequence before analysis as outlined below (for a review, see Abramson et al., Current Opinion in Biotechnology, 4:41-47 (1993)).
Sensitivity, i.e. detection limits, remain a significant obstacle in nucleic acid detection systems, and a variety of techniques have been developed to address this issue. Briefly, these techniques can be classified as either target amplification or signal amplification. Target amplification involves the amplification (i.e. replication) of the target sequence to be detected, resulting in a significant increase in the number of target molecules. Target amplification strategies include the polymerase chain reaction (PCR), strand displacement amplification (SDA), nucleic acid sequence based amplification (NASBA), and transcription mediated amplification (TMA).
Alternatively, rather than amplify the target, alternate techniques use the target as a template to replicate a signalling probe, allowing a small number of target molecules to result in a large number of signalling probes, that then can be detected. Signal amplification strategies include the ligase chain reaction (LCR), cycling probe technology (CPT), Invader(trademark), Q-beta replicase (QBR), and the use of xe2x80x9camplification probesxe2x80x9d such as xe2x80x9cbranched DNAxe2x80x9dthat result in multiple label probes binding to a single target sequence.
The polymerase chain reaction (PCR) is widely used and described, and involve the use of primer extension combined with thermal cycling to amplify a target sequence; see U.S. Pat. Nos. 4,683,195 and 4,683,202, and PCR Essential Data, J. W. Wiley and sons, Ed. C. R. Newton, 1995, all of which are incorporated by reference. In addition, there are a number of variations of PCR which may also find use in the invention, including xe2x80x9cquantitative competitive PCRxe2x80x9dor xe2x80x9cQC-PCRxe2x80x9d, xe2x80x9carbitrarily primed PCRxe2x80x9d or xe2x80x9cAP-PCRxe2x80x9d, xe2x80x9cimmuno-PCRxe2x80x9d, xe2x80x9cAlu-PCRxe2x80x9d, xe2x80x9cPCR single strand conformational polymorphismxe2x80x9d or xe2x80x9cPCR-SSCPxe2x80x9d, xe2x80x9creverse transcriptase PCRxe2x80x9d or xe2x80x9cRT-PCRxe2x80x9d, xe2x80x9cbiotin capture PCRxe2x80x9d, xe2x80x9cvectorette PCRxe2x80x9d. xe2x80x9cpanhandle PCRxe2x80x9d, and xe2x80x9cPCR select cDNA subtrationxe2x80x9d, among others.
Strand displacement amplification (SDA) is generally described in Walker et al., in Molecular Methods for Virus Detection, Academic Press, Inc., 1995, and U.S. Pat. Nos. 5,455,166 and 5,130,238, all of which are hereby incorporated by reference.
Nucleic acid sequence based amplification (NASBA) is generally described in U.S. Pat. No. 5,409,818; Sooknanan et al., Nucleic Acid Sequence-Based Amplification, Ch. 12 (pp. 261-285) of Molecular Methods for Virus Detection, Academic Press, 1995; and xe2x80x9cProfiting from Gene-based Diagnosticsxe2x80x9d, CTB International Publishing Inc., N.J., 1996, both of which are incorporated by reference.
Transcription mediated amplification (TMA) is generally described in U.S. Pat. Nos. 5,399,491, 5,888,779, 5,705,365, 5,710,029, all of which are incorporated by reference.
Cycling probe technology (CPT) is a nucleic acid detection system based on signal or probe amplification rather than target amplification, such as is done in polymerase chain reactions (PCR). Cycling probe technology relies on a molar excess of labeled probe which contains a scissile linkage of RNA. Upon hybridization of the probe to the target, the resulting hybrid contains a portion of RNA:DNA. This area of RNA:DNA duplex is recognized by RNAseH and the RNA is excised, resulting in cleavage of the probe. The probe now consists of two smaller sequences which may be released, thus leaving the target intact for repeated rounds of the reaction. The unreacted probe is removed and the label is then detected. CPT is generally described in U.S. Pat. Nos. 5,011,769, 5,403,711, 5,660,988, and 4,876,187, and PCT published applications WO 95/05480, WO 95/1416, and WO 95/00667, all of which are specifically incorporated herein by reference.
The ligation chain reaction (LCR) involve the ligation of two smaller probes into a single long probe, using the target sequence as the template for the ligase. See generally U.S. Pat. Nos. 5,185,243 and 5,573,907; EP 0 320 308 B1; EP 0 336 731 B1; EP 0 439 182 B1; WO 90/01069; WO 89/12696; and WO 89/09835, all of which are incorporated by reference.
Q-beta replicase (QBR) is a mRNA amplification technique, similar to NASBA and TMA, that relies on an RNA-dependent RNA polymerase derived from the bacteriophage Q-beta that can synthesize up to a billion stands of product from a template.
Invader(trademark) technology is based on structure-specific polymerases that cleave nucleic acids in a site-specific manner. Two probes are used: an xe2x80x9cinvaderxe2x80x9d probe and a xe2x80x9csignallingxe2x80x9d probe, that adjacently hybridize to a target sequence with a non-complementary overlap. The enzyme cleaves at the overlap due to its recognition of the xe2x80x9ctailxe2x80x9d, and releases the xe2x80x9ctailxe2x80x9d with a label. This can then be detected. The Invader(trademark) technology is described in U.S. Pat. Nos. 5,846,717; 5,614,402; 5,719,028; 5,541,311; and 5,843,669, all of which are hereby incorporated by reference.
xe2x80x9cRolling circle amplificationxe2x80x9d is based on extension of a circular probe that has hybridized to a target sequence. A polymerase is added that extends the probe sequence. As the circular probe has no terminus, the polymerase repeatedly extends the circular probe resulting in concatamers of the circular probe. As such, the probe is amplified. Rolling-circle amplification is generally described in Baner et al. (1998) Nuc. Acids Res. 26:5073-5078; Barany, F. (1991) Proc. Natl. Acad. Sci. USA 88:189-193; Lizardi et al. (1998) Nat. Genet. 19:225-232; Zhang et al., Gene 211:277 (1998); and Daubendiek et al., Nature Biotech. 15:273 (1997); all of which are incorporated by reference in their entirety.
xe2x80x9cBranched DNAxe2x80x9d signal amplification relies on the synthesis of branched nucleic acids, containing a multiplicity of nucleic acid xe2x80x9carmsxe2x80x9d that function to increase the amount of label that can be put onto one probe. This technology is generally described in U.S. Pat. Nos. 5,681,702, 5,597,909, 5,545,730, 5,594,117, 5,591,584, 5,571,670, 5,580,731, 5,571,670, 5,591,584, 5,624,802, 5,635,352, 5,594,118, 5,359,100, 5,124,246 and 5,681,697, all of which are hereby incorporated by reference.
Similarily, dendrimers of nucleic acids serve to vastly increase the amount of label that can be added to a single molecule, using a similar idea but different compositions. This technology is as described in U.S. Pat. No. 5,175,270 and Nilsen et al., J. Theor. Biol. 187:273 (1997), both of which are incorporated herein by reference.
Finally, U.S. Pat. Nos. 5,591,578, 5,824,473, 5,770,369, 5,705,348, and 5,780,234, and PCT application WO098/20162 describe novel compositions comprising nucleic acids containing electron transfer moieties, including electrodes, which allow for novel detection methods of nucleic acid hybridization.
In accordance with the objects outlined above, the present invention provides methods for detecting a target nucleic acid sequence. The methods comprise hybridizing at least a first primer nucleic acid to the target sequence to form a first hybridization complex, and contacting the first hybridization complex with a first enzyme to form a modified first primer nucleic acid. The first hybridization complex is then disassociated. These steps may be repeated a plurality of times. A first assay complex is then formed comprising at least one ETM and the modified first primer nucleic acid. The assay complex is covalently attached to an electrode. Electron transfer is then detected between the ETM and the electrode as an indication of the presence of the target sequence. The method can include the same method on a second target sequence substantially complementary to the the first target sequence.
In an additional aspect, the method utilizes a DNA polymerase and the modification to the primer is an extension of the primer such that the polymerase chain reaction (PCR) occurs.
In a further aspect, the method utilizes a ligase and the modification to the primer comprises a ligation of the first primer which hybridizes to a first domain of the first target sequence to a third primer which hybridizes to a second adjacent domain of the first target sequence, such that the ligase chain reaction (LCR) occurs.
In an additional aspect, the method utilizes a first primer comprising a first probe sequence, a first scissile linkage and a second probe sequence. The enzyme will cleave the first scissile linkage resulting in the separation of the first and the second probe sequences and the disassociation of the hybridization complex while leaving the first target sequence intact, such that the cycling probe technology (CPT) reaction occurs.
In a further aspect, the method utilizes a first enzyme that is a polymerase that extends the first primer to form a modified first primer comprising a first newly synthesized strand, and said method further comprises the addition of a second enzyme comprising a nicking enzyme that nicks the extended first primer leaving the first target sequence intact. The method additionally comprises extending from the nick using the polymerase, thereby displacing the first newly synthesized strand and generating a second newly synthesized strand, such that strand displacement amplification (SDA) occurs.
In an additional aspect, the method utilizes a first target sequence that is a RNA target sequence, a first primer nucleic acid that is a DNA primer comprising an RNA polymerase promoter, and the first enzyme is a reverse-transcriptase that extends the first primer to form a first newly synthesized DNA strand. The method further comprises the addition of a second enzyme comprising an RNA degrading enzyme that degrades the first target sequence. A third primer is then added that hybridizes to the first newly synthesized DNA strand. A third enzyme is added comprising a DNA polymerase that extends the third primer to form a second newly synthesized DNA strand, to form a newly synthesized DNA hybrid. A fourth enzyme is then added comprising an RNA polymerase that recognizes the RNA polymerase promoter and generates at least one newly synthesized RNA strand from the DNA hybrid, such that nucleic acid sequence-based amplification (NASBA) occurs.
In a further aspect, the invention provides methods for detecting a target nucleic acid sequence comprising forming a first hybridization complex comprising an amplifier probe and a target sequence, wherein the amplifier probe comprises at least two amplification sequences and hybridizing a first portion of at least one label probe to all or part of at least one amplification sequence. A second portion of the label probe is then hybridized to a detection probe covalently attached to an electrode via a conductive oligomer to form a second hybridization complex that contains at least a first electron transfer moiety (ETM). The label probe is then detected by measuring electron transfer between said first ETM and said electrode.
In an additional aspect, the invention provides methods for detecting a target nucleic acid sequence comprising forming a first hybridization complex comprising an amplifier probe and a target sequence, wherein the amplifier probe comprises at least two amplification sequences and wherein the first hybridization complex is covalently attached to an electrode comprising a monolayer comprising conductive oligomers. At least one label probe comprising at least one electron transfer moiety (ETM) is hybridized to all or part of at least one amplification sequence, and the label probe is detected by measuring electron transfer between said first ETM and said electrode.
In a further aspect, the invention provides kits for the detection of a first target nucleic acid sequence comprising at least a first nucleic acid primer substantially complementary to at least a first domain of the target sequence and at least a first enzyme that will modify the first nucleic acid primer. The kits additionally comprise an electrode comprising at least one detection probe covalently attached to the electrode via a conductive oligomer.
In an additional aspect, the invention provides methods of detecting target sequences comprising providing a rolling circle probe (RCP) comprising a first ligation sequence substantially complementary to a first domain of said target sequence, a second ligation sequence substantially complementary to a second domain of said target sequence; and a priming sequence. The methods further comprise hybridizing the first ligation sequence to said first domain and the second ligation sequence to the second domain to form a first hybridization complex and ligating the first and second ligation sequences together. A primer substantially complementary to said priming sequence, a polymerase, dNTPs and an ETM are added to the first hybridization complex under conditions whereby a rolling circle concatamer is formed, and the ETM is detected as an indicator of the presence of the target sequence. The RCP may further optionally comprise a cleavage site and a capture sequence.
In a further aspect, the invention provides methods for detecting a first target nucleic acid sequence comprising hybridizing an invader primer and a signaling primer to form a first hybridization complex. The signaling primer comprises a first portion comprising a sequence that will hybridize to a first portion of the target sequence; a cleavage site and a detection sequence that does not hybridize with the target sequence. The first hybridization complex is contacted with a structure specific cleavage enzyme such that the signaling primer is cleaved and the detection sequence is released. The released detection sequence is contacted with an electrode comprising a capture probe to form a second hybridization complex, wherein the second hybridization complex comprises at least one ETM. The ETM is detected as an indication of the presence of said target sequence.